479 results on '"Malate synthase"'
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2. The Glyoxylate Shunt, 60 Years On.
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Dolan, Stephen K. and Welch, Martin
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2017 marks the 60th anniversary of Krebs’ seminal paper on the glyoxylate shunt (and coincidentally, also the 80th anniversary of his discovery of the citric acid cycle). Sixty years on, we have witnessed substantial developments in our understanding of how flux is partitioned between the glyoxylate shunt and the oxidative decarboxylation steps of the citric acid cycle. The last decade has shown us that the beautifully elegant textbook mechanism that regulates carbon flux through the shunt in E. coli is an oversimplification of the situation in many other bacteria. The aim of this review is to assess how this new knowledge is impacting our understanding of flux control at the TCA cycle/glyoxylate shunt branch point in a wider range of genera, and to summarize recent findings implicating a role for the glyoxylate shunt in cellular functions other than metabolism. [ABSTRACT FROM AUTHOR]
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- 2018
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3. Monoterpenoid perillyl alcohol impairs metabolic flexibility of Candida albicans by inhibiting glyoxylate cycle.
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Ansari, Moiz A., Fatima, Zeeshan, Ahmad, Kamal, and Hameed, Saif
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MONOTERPENOIDS , *CANDIDA albicans , *MALATE synthase , *BINDING energy , *DISEASE prevalence - Abstract
The metabolic pathway such as glyoxylate cycle (GC) enables Candida albicans, to survive under glucose deficient conditions prevalent in the hostile niche. Thus its key enzymes (Isocitrate lyase; ICL and malate synthase; MLS) represent attractive targets against C. albicans . We have previously reported the antifungal potential of a natural monoterpenoid perillyl alcohol (PA). The present study uncovers additional role of PA as a potent GC inhibitor. We explored that PA phenocopied ICL1 deletion mutant and were hypersensitive under low carbon utilizing conditions. The effect of PA on GC was substantiated by molecular docking analyses, which reveals the in-silico binding affinity of PA with ICL and MLS and explored that PA binds to the active sites of both proteins with better binding energy in comparison to their known inhibitors 3-nitropropionate and bromopyruvate respectively. Enzyme kinetics by Lineweaver-Burk plot unravels that PA inhibits ICL and MLS enzymes in competitive and non-competitive manner respectively. Moreover, semi-quantitative RT-PCR indicated that PA inhibits ICL1 and MLS1 mRNA expressions. Lastly, we demonstrated the antifungal efficacy of PA by enhanced survival of Caenorhabditis elegans model and less hemolytic activity (10.6%) on human blood cells. Further studies are warranted for PA to be considered as viable drug candidate. [ABSTRACT FROM AUTHOR]
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- 2018
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4. Malate synthase contributes to the survival of Salmonella Typhimurium against nutrient and oxidative stress conditions
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Ratanti, Sarkhel, Shekhar, Apoorva, Swagatika, Priyadarsini, Hari Balaji, Sridhar, Sanjeev Kumar, Bhure, and Manish, Mahawar
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Salmonella typhimurium ,Oxidative Stress ,Glucose ,Multidisciplinary ,Malate Synthase ,Glyoxylates ,Nutrients ,Acetates ,Carbon Dioxide ,Isocitrate Lyase ,Carbon ,Hypochlorous Acid - Abstract
To survive and replicate in the host, S. Typhimurium have evolved several metabolic pathways. The glyoxylate shunt is one such pathway that can utilize acetate for the synthesis of glucose and other biomolecules. This pathway is a bypass of the TCA cycle in which CO2 generating steps are omitted. Two enzymes involved in the glyoxylate cycle are isocitrate lyase (ICL) and malate synthase (MS). We determined the contribution of MS in the survival of S. Typhimurium under carbon limiting and oxidative stress conditions. The ms gene deletion strain (∆ms strain) grew normally in LB media but failed to grow in M9 minimal media supplemented with acetate as a sole carbon source. However, the ∆ms strain showed hypersensitivity (p ms strain has been significantly more susceptible to neutrophils. Interestingly, several folds induction of ms gene was observed following incubation of S. Typhimurium with neutrophils. Further, ∆ms strain showed defective colonization in poultry spleen and liver. In short, our data demonstrate that the MS contributes to the virulence of S. Typhimurium by aiding its survival under carbon starvation and oxidative stress conditions.
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- 2022
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5. Synthesis and Function of Glyoxylate Cycle Enzymes
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Cornah, Johanna E., Smith, Steven M., Baker, Alison, editor, and Graham, Ian A., editor
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- 2002
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6. Gene expression profiling of protease and non-protease genes in Trichophyton mentagrophytes isolates from dermatophytosis patients by qRT-PCR analysis
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Shyama Datt, Sambit Nath Bhattacharya, Thakur Datt, Shukla Das, M. Ahmad Ansari, and Rahul Sharma
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0301 basic medicine ,Antigens, Fungal ,Virulence Factors ,medicine.medical_treatment ,Science ,030106 microbiology ,Genes, Fungal ,Glyoxylate cycle ,Virulence ,India ,Pathogenesis ,Microbiology ,Article ,Applied microbiology ,03 medical and health sciences ,Tinea ,Malate synthase ,Gene Expression Regulation, Fungal ,medicine ,Humans ,DNA, Fungal ,Gene ,chemistry.chemical_classification ,Metalloproteinase ,Multidisciplinary ,Protease ,biology ,Reverse Transcriptase Polymerase Chain Reaction ,Arthrodermataceae ,Gene Expression Profiling ,Fungi ,Isocitrate lyase ,Microarray Analysis ,030104 developmental biology ,Enzyme ,chemistry ,biology.protein ,Medicine ,Peptide Hydrolases - Abstract
Trichophyton mentagrophytes secretes Metallocarboxypeptidase A and B of the M14 family as endoproteases and exoprotease. T. mentagrophytes produce Metalloprotease 3 and 4 which degrades the protein into the short peptides and amino acids. To understand the host fungal relationship and identification of such genes expressed during infection is utmost important. T. mentagrophytes encodes some proteins which are associated with the glyoxylate cycle. The glyoxylate cycle enzymes have been involving in virulence of dermatophytes and their up-regulation during dermatophytes growth on keratin. On comparing the expression level of virulence protease and non-protease genes, we observed, among exoprotease protease genes, Metallocarboxypeptidase B was strongly up regulated (134.6 fold high) followed by Metallocarboxypeptidase A (115.6 fold high) and Di-peptidyl-peptidases V (10.1 fold high), in dermatophytic patients as compared to ATCC strain. Furthermore, among endoprotease, Metalloprotease 4 was strongly up regulated (131.6 fold high) followed by Metalloprotease 3 (16.7 fold high), in clinical strains as compared to T. mentagrophytes ATCC strain. While among non-protease genes, Citrate Synthase was highly expressed (118 fold high), followed by Isocitrate Lyase (101.6 fold high) and Malate Synthase (52.9 fold high). All the studied virulence genes were considered the best suitable ones by geNorm, Best keeper, Norm Finder and Ref finder.
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- 2021
7. Apparent isocitrate lyase activity in Leishmania amazonensis.
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Hernández-Chinea, Concepción, Maimone, Laura, Campos, Yelitza, Mosca, Walter, and Romero, Pedro J.
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LEISHMANIA ,ISOCITRATE lyase ,MALATE synthase ,PROTOZOA genetics ,SPECTROPHOTOMETRY ,IMMUNOBLOTTING - Abstract
Early reports have demonstrated the occurrence of glyoxylate cycle enzymes in several Leishmania species. However, these results have been underestimated because genes for the two key enzymes of the cycle, isocitrate lyase (ICL) and malate synthase (MS), are not annotated in Leishmania genomes. We have re-examined this issue in promastigotes of Leishmania amazonensis. Enzyme activities were assayed spectrophotometrically in cellular extracts and characterized partially. A 40 kDa band displaying ICL activity was visualized on zymograms of the extracts. By immunoblotting with mouse antibodies against ICL from Bacillus stearothermophilus, a band of approximately 40 kDa was identified, coincident with the relative molecular mass of the activity band revealed on zymograms. Indirect immunofluorescence of intact promastigotes showed that the recognized antigen is distributed as a punctuated pattern, mainly distributed beneath the subpellicular microtubules, over a diffused cytoplasmic stain. These results clearly demonstrate the existence of an apparent ICL activity in L. amazonensis promastigotes, which is associated to a 40 kDa polypeptide and distributed both diffused and as punctuate aggregates in the cytoplasm. The relevance of this activity is discussed. [ABSTRACT FROM AUTHOR]
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- 2017
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8. Glyoxylate detoxification is an essential function of malate synthase required for carbon assimilation in Mycobacterium tuberculosis.
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Puckett, Susan, Trujillo, Carolina, Zhe Wang, Eoh, Hyungjin, Ioerger, Thomas R., Krieger, Inna, Sacchettini, James, Schnappinger, Dirk, Rhee, Kyu Y., and Ehrt, Sabine
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FATTY acids , *MYCOBACTERIUM tuberculosis , *ISOCITRATE lyase , *PHYSIOLOGY , *LUNG diseases - Abstract
The glyoxylate shunt is a metabolic pathway of bacteria, fungi, and plants used to assimilate even-chain fatty acids (FAs) and has been implicated in persistence of Mycobacterium tuberculosis (Mtb). Recent work, however, showed that the first enzyme of the glyoxylate shunt, isocitrate lyase (ICL), may mediate survival of Mtb during the acute and chronic phases of infection in mice through physiologic functions apart from fatty acid metabolism. Here, we report that malate synthase (MS), the second enzyme of the glyoxylate shunt, is essential for in vitro growth and survival of Mtb on even-chain fatty acids, in part, for a previously unrecognized activity: mitigating the toxicity of glyoxylate excess arising from metabolism of even-chain fatty acids. Metabolomic profiling revealed that MS-deficient Mtb cultured on fatty acids accumulated high levels of the ICL aldehyde endproduct, glyoxylate, and increased levels of acetyl phosphate, acetoacetyl coenzyme A (acetoacetyl-CoA), butyryl CoA, acetoacetate, and β-hydroxybutyrate. These changes were indicative of a glyoxylate- induced state of oxaloacetate deficiency, acetate overload, and ketoacidosis. Reduction of intrabacterial glyoxylate levels using a chemical inhibitor of ICL restored growth ofMS-deficient Mtb, despite inhibiting entry of carbon into the glyoxylate shunt. In vivo depletion of MS resulted in sterilization of Mtb in both the acute and chronic phases of mouse infection. This work thus identifies glyoxylate detoxification as an essential physiologic function of Mtb malate synthase and advances its validation as a target for drug development. [ABSTRACT FROM AUTHOR]
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- 2017
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9. Genome Scale Metabolic Model of the versatile methanotroph Methylocella silvestris
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Andrew T. Crombie, Sergio Bordel, Raúl Muñoz, and J. Colin Murrell
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Proteomics ,Methanotroph ,Glyoxylate cycle ,lcsh:QR1-502 ,Bioengineering ,Computational biology ,Models, Biological ,Applied Microbiology and Biotechnology ,lcsh:Microbiology ,Metabolic engineering ,Industrial Microbiology ,Propane ,03 medical and health sciences ,Beijerinckiaceae ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Ethanol ,biology ,030306 microbiology ,Chemistry ,Methylocella silvestris ,Research ,Malate Synthase ,Glyoxylates ,Assimilation (biology) ,biology.organism_classification ,Isocitrate Lyase ,Carbon ,Metabolic pathway ,Enzyme ,Genes, Bacterial ,Mutation ,Genetic Engineering ,Energy source ,Methane ,Metabolic Networks and Pathways ,Biotechnology - Abstract
Background Methylocella silvestris is a facultative aerobic methanotrophic bacterium which uses not only methane, but also other alkanes such as ethane and propane, as carbon and energy sources. Its high metabolic versatility, together with the availability of tools for its genetic engineering, make it a very promising platform for metabolic engineering and industrial biotechnology using natural gas as substrate. Results The first Genome Scale Metabolic Model for M. silvestris is presented. The model has been used to predict the ability of M. silvestris to grow on 12 different substrates, the growth phenotype of two deletion mutants (ΔICL and ΔMS), and biomass yield on methane and ethanol. The model, together with phenotypic characterization of the deletion mutants, revealed that M. silvestris uses the glyoxylate shuttle for the assimilation of C1 and C2 substrates, which is unique in contrast to published reports of other methanotrophs. Two alternative pathways for propane metabolism have been identified and validated experimentally using enzyme activity tests and constructing a deletion mutant (Δ1641), which enabled the identification of acetol as one of the intermediates of propane assimilation via 2-propanol. The model was also used to integrate proteomic data and to identify key enzymes responsible for the adaptation of M. silvestris to different substrates. Conclusions The model has been used to elucidate key metabolic features of M. silvestris, such as its use of the glyoxylate shuttle for the assimilation of one and two carbon compounds and the existence of two parallel metabolic pathways for propane assimilation. This model, together with the fact that tools for its genetic engineering already exist, paves the way for the use of M. silvestris as a platform for metabolic engineering and industrial exploitation of methanotrophs.
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- 2020
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10. Efficiency of vanillin in impeding metabolic adaptability and virulence of Candida albicans by inhibiting glyoxylate cycle, morphogenesis, and biofilm formation
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Fatima Zeeshan, Hameed Saif, Saibabu Venkata, Ahmad Kamal, and Ahmad Khan Luqman
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lcsh:Internal medicine ,biology ,Glyoxylate cycle ,Biofilm ,Virulence ,morphogenesis ,Isocitrate lyase ,biology.organism_classification ,Microbiology ,Corpus albicans ,biofilm ,Metabolic pathway ,Infectious Diseases ,caenorhabditis elegans ,vanillin ,lcsh:Biology (General) ,Malate synthase ,glyoxylate cycle ,biology.protein ,candida ,Original Article ,Candida albicans ,lcsh:RC31-1245 ,lcsh:QH301-705.5 - Abstract
Background and Purpose: Candida albicans is the fourth most common cause of nosocomial fungal infections across the world. The current drug regimens are suffering from such drawbacks as drug resistance, toxicity, and costliness; accordingly, they highlight the need for the discovery of novel drug agents. The metabolic adaptability under low-carbon conditions and expression of functional virulence traits mark the success of pathogens to cause infection. The metabolic pathways, such as glyoxylate cycle (GC), enable C. albicans to survive under glucose-deficient conditions prevalent in the hostile niche. Therefore, the key enzymes, namely isocitrate lyase (ICL) and malate synthase (MLS), represent attractive agents against C. albicans. Similarly, virulence traits, such as morphogenesis and biofilm formation, are the crucial determinants of C. albicans pathogenicity. Regarding this, the present study was conducted to uncover the role of vanillin (Van), a natural food flavoring agent, in inhibiting GC, yeast-to-hyphal transition, and biofilm formation in human fungal pathogen C. albicans. Materials and Methods: For the determination of hypersensitivity under low-glucose conditions, phenotypic susceptibility assay was utilized. In addition, enzyme activities were estimated based on crude extracts while in-silico binding was confirmed by molecular docking. The assessment of morphogenesis was accomplished using hyphalinducing media, and biofilm formation was estimated using calcofluor staining, MTT assay, and biomass measurement. Additionally, the in vivo efficacy of Van was demonstrated using Caenorhabditis elegans nematode model. Results: Based on the results, Van was found to be a potent GC inhibitor that phenocopied ICL1 deletion mutant and displayed hypersensitivity under low-carbon conditions. Accordingly, Van facilitated the inhibition of ICL and MLS activities in vitro. Molecular docking analyses revealed the in-silico binding affinity of Van with Icl1p and Mls1p. Those analyses were also confirmative of the binding of Van to the active sites of both proteins with better binding energy in comparison to their known inhibitors. Furthermore, Van led to the attenuation of such virulence traits as morphogenesis, biofilm formation, and cell adherence. Finally, the antifungal efficacy of Van was demonstrated by the enhanced survival of C. elegans with Candida infection. The results also confirmed negligible hemolytic activity on erythrocytes. Conclusion: As the findings of the present study indicated, Van is a persuasive natural compound that warrants further attention to exploit its anticandidal potential
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- 2020
11. Role of Glyoxylate Shunt in Oxidative Stress Response.
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Ahn, Sungeun, Jaejoon Jung, In-Ae Jang, Madsen, Eugene L., and Park, Woojun
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KREBS cycle , *OXIDATIVE stress , *ISOCITRATE lyase , *MALATE synthase , *ACID metabolism , *PSEUDOMONAS aeruginosa , *ESCHERICHIA coli - Abstract
The glyoxylate shunt (GS) is a two-step metabolic pathway (isocitrate lyase, aceA; and malate synthase, glcB) that serves as an alternative to the tricarboxylic acid cycle. The GS bypasses the carbon dioxide-producing steps of the tricarboxylic acid cycle and is essential for acetate and fatty acid metabolism in bacteria. GS can be up-regulated under conditions of oxidative stress, antibiotic stress, and host infection, which implies that it plays important but poorly explored roles in stress defense and pathogenesis. In many bacterial species, including Pseudomonas aeruginosa, aceA and glcB are not in an operon, unlike in Escherichia coli. In P. aeruginosa, we explored relationships between GS genes and growth, transcription profiles, and biofilm formation. Contrary to our expectations, deletion of aceA in P. aeruginosa improved cell growth under conditions of oxidative and antibiotic stress. Transcriptome data suggested that aceA mutants underwent a metabolic shift toward aerobic denitrification; this was supported by additional evidence, including up-regulation of denitrification-related genes, decreased oxygen consumption without lowering ATP yield, increased production of denitrification intermediates (NO and N2O), and increased cyanide resistance. The aceA mutants also produced a thicker exopolysaccharide layer; that is, a phenotype consistent with aerobic denitrification. A bioinformatic survey across known bacterial genomes showed that only microorganisms capable of aerobic metabolism possess the glyoxylate shunt. This trend is consistent with the hypothesis that the GS plays a previously unrecognized role in allowing bacteria to tolerate oxidative stress. [ABSTRACT FROM AUTHOR]
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- 2016
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12. Effect of Target Gene Silencing on Calcite Single Crystal Formation by Thermophilic Bacterium Geobacillus thermoglucosidasius NY05
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Keiji Kiyoshi, Naoto Yoshida, and Rie Murai
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Glyoxylate cycle ,Acetates ,Applied Microbiology and Biotechnology ,Microbiology ,Calcium Carbonate ,03 medical and health sciences ,chemistry.chemical_compound ,Bacterial Proteins ,Geobacillus thermoglucosidasius ,Malate synthase ,Gene Silencing ,030304 developmental biology ,chemistry.chemical_classification ,Calcite ,0303 health sciences ,DNA ligase ,biology ,030306 microbiology ,Oligonucleotide ,Thermophile ,Malate Synthase ,Geobacillus ,Glyoxylates ,General Medicine ,Isocitrate lyase ,biology.organism_classification ,Isocitrate Lyase ,Biochemistry ,chemistry ,biology.protein ,Calcium - Abstract
Geobacillus thermoglucosidasius NY05 catalyzes calcite single crystal formation at 60 °C by using acetate and calcium. Endospores are embedded at the central part of the calcite single crystal and carbon atoms in the calcite lattice are derived from acetate carbon. Here, we synthesized 21-mer antisense DNA oligonucleotides targeting sporulation transcription factor, acetate-CoA ligase, isocitrate lyase, and malate synthase G mRNAs and evaluated the effect of these oligonucleotides on calcite formation in G. thermoglucosidasius NY05. G. thermoglucosidasius NY05 cells containing antisense DNA oligonucleotides targeting sporulation transcription factor, acetate-CoA ligase, isocitrate lyase, and malate synthase G mRNAs had reduced calcite single crystal formation by 18.7, 50.6, 55.7, and 82.3%, respectively, compared with cells without antisense DNA oligonucleotides. These results support that calcite formation needs endospores as the nucleus to grow, and carbon dioxide generated from acetate, which is metabolized via the glyoxylate pathway and glucogenesis, is supplied to the crystal lattice.
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- 2019
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13. Glyoxylate cycle activity in Pinus pinea seeds during germination in altered gravity conditions
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Paola Faraoni, Elettra Sereni, Francesco Ranaldi, Francesca Cialdai, Monica Monici, and A. Gnerucci
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0106 biological sciences ,0301 basic medicine ,Physiology ,Glyoxylate cycle ,Germination ,Plant Science ,01 natural sciences ,03 medical and health sciences ,Malate synthase ,Genetics ,Radicle ,Hypergravity ,biology ,Weightlessness ,Chemistry ,Glyoxylates ,Isocitrate lyase ,Pinus ,Citric acid cycle ,Metabolic pathway ,Horticulture ,030104 developmental biology ,Seeds ,biology.protein ,010606 plant biology & botany - Abstract
This work inserts in the research field regarding the effects of altered gravity conditions on biological plant processes. Pinus pinea seeds germination was studied in simulated microgravity (2x10−3g) and hypergravity (20g) conditions. The effects of simulated gravity were evaluated monitoring the levels of the key enzymes, involved in the main metabolic pathway during germination process of lipid–rich seeds (oilseeds): isocitrate lyase and malate synthase for glyoxylate cycle, 3-hydroxyacyl-CoA dehydrogenase for beta-oxidation, isocitrate dehydrogenase for Krebs cycle, pyruvate kinase for glycolysis and glucose 6 phosphate dehydrogenase for pentose phosphate shunt. The simulated micro and hypergravity conditions were obtained by a Random Position Machine and a Hyperfuge, respectively. Results show that the levels of some tested enzymes, at different lag times of the germination process, have the same trend of controls (g = 1), but with significant differences from quantitative point of view. They are higher in microgravity conditions and lower in hypergravity ones, suggesting that, from a biochemical point of view, the germination process results accelerated in microgravity conditions and delayed in hypergravity ones. These biochemical results show a good correlation with morphological ones, obtained with the measurement of the length of the seeds sprouting radicle. These results give promising indications regarding the possibility to grow plant with lipid-rich seeds in spatial environment, to obtain food sources for astronauts during long term space missions and to reconstitute new atmosphere.
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- 2019
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14. Metabolic fitness of
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Zeeshan Fatima, Saif Hameed, and Sandeep Hans
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QH301-705.5 ,Glyoxylate cycle ,chitin ,Microbiology ,chemistry.chemical_compound ,Chitin ,Malate synthase ,glyoxylate cycle ,Biology (General) ,Candida albicans ,Internal medicine ,Ergosterol ,ergosterol ,biology ,Isocitrate lyase ,biology.organism_classification ,RC31-1245 ,Metabolic pathway ,Infectious Diseases ,chemistry ,Biochemistry ,candida ,biology.protein ,efflux pump ,Original Article ,Efflux - Abstract
Background and Purpose: The increment in fungal infections, particularly due to Candida species, is alarming due to the emergence of multidrug resistance (MDR). Hence, the identification of novel drug targets to circumvent the problem of MDR requires immediate attention. The metabolic pathway, such as glyoxylate cycle (GC), which utilizes key enzymes (isocitrate lyase [ICL] and malate synthase [MLS]), enables C. albicans to adapt under glucose-deficient conditions. This study uncovers the effect of GC disruption on the major MDR mechanisms of C. albicans as a human pathogenic fungus. Materials and Methods: For the purpose of the study, efflux pump activity was assessed by phenotypic susceptibilities in the presence of substrates rhodamine 6G (R6G) and Nile red, along with R6G extracellular concentration (527 nm). In addition, ergosterol content was estimated by the alcoholic potassium hydroxide hydrolysis method. The estimation of chitin was also accomplished by the absorbance (520 nm) of glucosamine released by acid hydrolysis. Results: The results revealed that the disruption of ICL enzyme gene (Δicl1) led to the impairment of the efflux activity of multidrug transporters belonging to the ATP-binding cassette superfamily. It was further shown that Δicl1 mutant exhibited diminished ergosterol and chitin contents. In addition, all abrogated phenotypes could be rescued in the reverting strain of Δicl1 mutant. Conclusion: Based on the findings, the disruption of GC affected efflux activity and the synthesis of ergosterol and chitin. The present study for the first time revealed that metabolic fitness was associated with functional drug efflux, ergosterol and chitin biosynthesis and validated GC as an antifungal target. However, further studies are needed to comprehend and exploit this therapeutic opportunity.
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- 2021
15. Production of succinate by engineered strains of Synechocystis PCC 6803 overexpressing phosphoenolpyruvate carboxylase and a glyoxylate shunt
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Alessia Albergati, Jeffrey A. Hawkes, Kateryna Kukil, Peter Lindblad, Pia Lindberg, and Claudia Durall
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Succinate ,Glyoxylate cycle ,lcsh:QR1-502 ,Succinic Acid ,Bioengineering ,Microbiology ,Applied Microbiology and Biotechnology ,lcsh:Microbiology ,Synechocystis PCC 6803 ,2-thenoyltrifluoroacetone ,Bacterial Proteins ,Malate synthase ,Phosphoenolpyruvate carboxylase ,TCA cycle ,biology ,Chemistry ,Acetate ,Succinate dehydrogenase ,Research ,Synechocystis ,Glyoxylates ,Isocitrate lyase ,biology.organism_classification ,Citric acid cycle ,Mikrobiologi ,Biochemistry ,Metabolic Engineering ,Glyoxylate shunt ,biology.protein ,Heterologous expression ,Phosphoenolpyruvate Carboxykinase (ATP) ,Biotechnology - Abstract
Background Cyanobacteria are promising hosts for the production of various industrially important compounds such as succinate. This study focuses on introduction of the glyoxylate shunt, which is naturally present in only a few cyanobacteria, into Synechocystis PCC 6803. In order to test its impact on cell metabolism, engineered strains were evaluated for succinate accumulation under conditions of light, darkness and anoxic darkness. Each condition was complemented by treatments with 2-thenoyltrifluoroacetone, an inhibitor of succinate dehydrogenase enzyme, and acetate, both in nitrogen replete and deplete medium. Results We were able to introduce genes encoding the glyoxylate shunt, aceA and aceB, encoding isocitrate lyase and malate synthase respectively, into a strain of Synechocystis PCC 6803 engineered to overexpress phosphoenolpyruvate carboxylase. Our results show that complete expression of the glyoxylate shunt results in higher extracellular succinate accumulation compared to the wild type control strain after incubation of cells in darkness and anoxic darkness in the presence of nitrate. Addition of the inhibitor 2-thenoyltrifluoroacetone increased succinate titers in all the conditions tested when nitrate was available. Addition of acetate in the presence of the inhibitor further increased the succinate accumulation, resulting in high levels when phosphoenolpyruvate carboxylase was overexpressed, compared to control strain. However, the highest succinate titer was obtained after dark incubation of an engineered strain with a partial glyoxylate shunt overexpressing isocitrate lyase in addition to phosphoenolpyruvate carboxylase, with only 2-thenoyltrifluoroacetone supplementation to the medium. Conclusions Heterologous expression of the glyoxylate shunt with its central link to the tricarboxylic acid cycle (TCA) for acetate assimilation provides insight on the coordination of the carbon metabolism in the cell. Phosphoenolpyruvate carboxylase plays an important role in directing carbon flux towards the TCA cycle.
- Published
- 2021
16. Transcriptomic and proteomic profiling revealed reprogramming of carbon metabolism in acetate-grown human pathogen Candida glabrata
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Doblin Sandai, Benjamin Yii Chung Lau, Kok Lian Ho, Shu Yih Chew, H Yahaya, Leslie Thian Lung Than, Yoke Kqueen Cheah, and Alistair J. P. Brown
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0301 basic medicine ,Proteome ,Liquid chromatography tandem-mass spectrometry ,Endocrinology, Diabetes and Metabolism ,030106 microbiology ,Clinical Biochemistry ,RNA-sequencing ,Glyoxylate cycle ,lcsh:Medicine ,Candida glabrata ,Acetates ,Biology ,Microbiology ,Fungal Proteins ,03 medical and health sciences ,PCK1 ,Malate synthase ,Pharmacology (medical) ,Candida albicans ,Molecular Biology ,Candida ,Acetate ,Gene Expression Profiling ,Research ,lcsh:R ,Biochemistry (medical) ,Carbon metabolism ,Proteomic ,Cell Biology ,General Medicine ,Metabolism ,Isocitrate lyase ,biology.organism_classification ,Carbon ,030104 developmental biology ,Transcriptomic ,biology.protein ,Transcriptome ,Phosphoenolpyruvate carboxykinase - Abstract
Background Emergence of Candida glabrata, which causes potential life-threatening invasive candidiasis, has been widely associated with high morbidity and mortality. In order to cause disease in vivo, a robust and highly efficient metabolic adaptation is crucial for the survival of this fungal pathogen in human host. In fact, reprogramming of the carbon metabolism is believed to be indispensable for phagocytosed C. glabrata within glucose deprivation condition during infection. Methods In this study, the metabolic responses of C. glabrata under acetate growth condition was explored using high-throughput transcriptomic and proteomic approaches. Results Collectively, a total of 1482 transcripts (26.96%) and 242 proteins (24.69%) were significantly up- or down-regulated. Both transcriptome and proteome data revealed that the regulation of alternative carbon metabolism in C. glabrata resembled other fungal pathogens such as Candida albicans and Cryptococcus neoformans, with up-regulation of many proteins and transcripts from the glyoxylate cycle and gluconeogenesis, namely isocitrate lyase (ICL1), malate synthase (MLS1), phosphoenolpyruvate carboxykinase (PCK1) and fructose 1,6-biphosphatase (FBP1). In the absence of glucose, C. glabrata shifted its metabolism from glucose catabolism to anabolism of glucose intermediates from the available carbon source. This observation essentially suggests that the glyoxylate cycle and gluconeogenesis are potentially critical for the survival of phagocytosed C. glabrata within the glucose-deficient macrophages. Conclusion Here, we presented the first global metabolic responses of C. glabrata to alternative carbon source using transcriptomic and proteomic approaches. These findings implicated that reprogramming of the alternative carbon metabolism during glucose deprivation could enhance the survival and persistence of C. glabrata within the host.
- Published
- 2021
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17. Catalase Inhibition Affects Glyoxylate Cycle Enzyme Expression and Cellular Redox Control during the Functional Transition of Sunflower and Safflower Seedlings.
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Torres, Taffarel, Silva, Ivanice, Castro, Érika, Santos, Elizeu, Cunha, Rodrigo, and Lima, João
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KREBS cycle ,CATALASE ,OXIDATION-reduction reaction ,CELLULAR control mechanisms ,SUNFLOWER seed oil ,SAFFLOWER oil ,NATURAL resources - Abstract
Oilseed crops are an important natural resource because they can be used for food and renewable energy production. However, oilseed seedling establishment and vigor depend upon the capacity to overcome functional transition, a developmental stage characterized by the consumption of the remaining oil reserves, through β-oxidation and glyoxylate cycle, and the onset of autotrophic metabolism. The increased growth and the acclimation to full photosynthetic activity lead to production of reactive oxygen species and a reorganization of the cell antioxidant systems to achieve a new redox homeostasis. In the present study, catalase (CAT) was inhibited by 3-amino-1,2,4-triazole application during functional transition in sunflower and safflower seedlings to understand the effect of this antioxidant enzyme impairment on the mRNA expression of the glyoxylate cycle enzymes isocitrate lyase (ICL) and malate synthase (MLS), as well as the superoxide dismutase (SOD) activity and ascorbate peroxidase (APX) activity and expression. CAT inhibition led to significant seedling growth reduction and increases in HO content, SOD activity, and mRNA expression of CAT and APX in both species. However, APX activity was induced only in safflower plants. Additionally, ICL and MLS mRNA expressions were upregulated after 6 h of treatment when compared to the control values. These results indicate that under CAT impairment conditions, redox homeostasis at the functional transition phase was partially supported by the SOD and APX antioxidant systems to maintain the seedling photosynthetic establishment. [ABSTRACT FROM AUTHOR]
- Published
- 2014
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18. Expression regulation of MALATE SYNTHASE involved in glyoxylate cycle during protocorm development in Phalaenopsis aphrodite (Orchidaceae)
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Chieh Kai Liang, Ming Hsiang Chuang, Tian-Hsiang Huang, Chih Hsiung Fu, Zhong Jian Liu, Yu Yun Hsiao, Wen Chieh Tsai, Wan Lin Wu, Li Jun Chen, and Hsiang Chia Lu
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0106 biological sciences ,0301 basic medicine ,Plant molecular biology ,Glyoxylate cycle ,Gene Expression ,lcsh:Medicine ,Germination ,01 natural sciences ,Article ,Endosperm ,Phalaenopsis aphrodite ,03 medical and health sciences ,Murashige and Skoog medium ,Gene Expression Regulation, Plant ,Malate synthase ,Botany ,Orchidaceae ,Symbiosis ,lcsh:Science ,Regulation of gene expression ,Multidisciplinary ,biology ,lcsh:R ,Malate Synthase ,Glyoxylates ,Isocitrate lyase ,biology.organism_classification ,030104 developmental biology ,Seedlings ,Seeds ,biology.protein ,Carbohydrate Metabolism ,lcsh:Q ,Transcriptome ,Plant embryogenesis ,Transcription Factors ,010606 plant biology & botany - Abstract
Orchid (Orchidaceae) is one of the largest families in angiosperms and presents exceptional diversity in lifestyle. Their unique reproductive characteristics of orchid are attracted by scientist for centuries. One of the synapomorphies of orchid plants is that their seeds do not contain endosperm. Lipids are used as major energy storage in orchid seeds. However, regulation and mobilization of lipid usage during early seedling (protocorm) stage of orchid is not understood. In this study, we compared transcriptomes from developing Phalaenopsis aphrodite protocorms grown on 1/2-strength MS medium with sucrose. The expression of P. aphrodite MALATE SYNTHASE (PaMLS), involved in the glyoxylate cycle, was significantly decreased from 4 days after incubation (DAI) to 7 DAI. On real-time RT-PCR, both P. aphrodite ISOCITRATE LYASE (PaICL) and PaMLS were down-regulated during protocorm development and suppressed by sucrose treatment. In addition, several genes encoding transcription factors regulating PaMLS expression were identified. A gene encoding homeobox transcription factor (named PaHB5) was involved in positive regulation of PaMLS. This study showed that sucrose regulates the glyoxylate cycle during orchid protocorm development in asymbiotic germination and provides new insights into the transcription factors involved in the regulation of malate synthase expression.
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- 2020
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19. 2-Aminopyridine Analogs Inhibit Both Enzymes of the Glyoxylate Shunt in Pseudomonas aeruginosa
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Mahmud Kajbaf, Päivi Tammela, Viviana Gatta, David R. Spring, Sean Bartlett, Alyssa C. McVey, Annalisa Pellacani, Martin Welch, Division of Pharmaceutical Biosciences, Drug Research Program, Bioactivity Screening Group, Divisions of Faculty of Pharmacy, Tammela, Päivi [0000-0003-4697-8066], Spring, David R [0000-0001-7355-2824], Welch, Martin [0000-0003-3646-1733], Apollo - University of Cambridge Repository, and Spring, David R. [0000-0001-7355-2824]
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0301 basic medicine ,MECHANISM ,116 Chemical sciences ,Aminopyridines ,medicine.disease_cause ,01 natural sciences ,lcsh:Chemistry ,glyoxylate shunt ,Cytotoxicity ,lcsh:QH301-705.5 ,Spectroscopy ,chemistry.chemical_classification ,biology ,Molecular Structure ,Glyoxylates ,General Medicine ,3. Good health ,Computer Science Applications ,Anti-Bacterial Agents ,isothermal titration calorimetry ,Biochemistry ,Enzyme inhibitor ,317 Pharmacy ,Pseudomonas aeruginosa ,ANTIBIOTICS ,MALATE SYNTHASE ,Glyoxylate cycle ,enzyme inhibitor ,malate synthase G ,Calorimetry ,Catalysis ,Cell Line ,Inorganic Chemistry ,03 medical and health sciences ,Bacterial Proteins ,Malate synthase ,medicine ,Humans ,Physical and Theoretical Chemistry ,Molecular Biology ,010405 organic chemistry ,Organic Chemistry ,Metabolism ,Isocitrate lyase ,Gene Expression Regulation, Bacterial ,ACIDS ,isocitrate lyase ,0104 chemical sciences ,030104 developmental biology ,Enzyme ,chemistry ,lcsh:Biology (General) ,lcsh:QD1-999 ,conditionally essential target ,biology.protein - Abstract
Pseudomonas aeruginosa is an opportunistic pathogen responsible for many hospital-acquired infections. P. aeruginosa can thrive in diverse infection scenarios by rewiring its central metabolism. An example of this is the production of biomass from C2 nutrient sources such as acetate via the glyoxylate shunt when glucose is not available. The glyoxylate shunt is comprised of two enzymes, isocitrate lyase (ICL) and malate synthase G (MS), and flux through the shunt is essential for the survival of the organism in mammalian systems. In this study, we characterized the mode of action and cytotoxicity of structural analogs of 2-aminopyridines, which have been identified by earlier work as being inhibitory to both shunt enzymes. Two of these analogs were able to inhibit ICL and MS in vitro and prevented growth of P. aeruginosa on acetate (indicating cell permeability). Moreover, the compounds exerted negligible cytotoxicity against three human cell lines and showed promising in vitro drug metabolism and safety profiles. Isothermal titration calorimetry was used to confirm binding of one of the analogs to ICL and MS, and the mode of enzyme inhibition was determined. Our data suggest that these 2-aminopyridine analogs have potential as anti-pseudomonal agents.
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- 2020
20. 2-Aminopyridine Analogs Inhibit Both Enzymes of the Glyoxylate Shunt in
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Alyssa C, McVey, Sean, Bartlett, Mahmud, Kajbaf, Annalisa, Pellacani, Viviana, Gatta, Päivi, Tammela, David R, Spring, and Martin, Welch
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Molecular Structure ,Malate Synthase ,malate synthase G ,enzyme inhibitor ,Aminopyridines ,Glyoxylates ,Gene Expression Regulation, Bacterial ,Calorimetry ,isocitrate lyase ,Article ,Anti-Bacterial Agents ,Cell Line ,isothermal titration calorimetry ,glyoxylate shunt ,Bacterial Proteins ,Pseudomonas aeruginosa ,conditionally essential target ,Humans - Abstract
Pseudomonas aeruginosa is an opportunistic pathogen responsible for many hospital-acquired infections. P. aeruginosa can thrive in diverse infection scenarios by rewiring its central metabolism. An example of this is the production of biomass from C2 nutrient sources such as acetate via the glyoxylate shunt when glucose is not available. The glyoxylate shunt is comprised of two enzymes, isocitrate lyase (ICL) and malate synthase G (MS), and flux through the shunt is essential for the survival of the organism in mammalian systems. In this study, we characterized the mode of action and cytotoxicity of structural analogs of 2-aminopyridines, which have been identified by earlier work as being inhibitory to both shunt enzymes. Two of these analogs were able to inhibit ICL and MS in vitro and prevented growth of P. aeruginosa on acetate (indicating cell permeability). Moreover, the compounds exerted negligible cytotoxicity against three human cell lines and showed promising in vitro drug metabolism and safety profiles. Isothermal titration calorimetry was used to confirm binding of one of the analogs to ICL and MS, and the mode of enzyme inhibition was determined. Our data suggest that these 2-aminopyridine analogs have potential as anti-pseudomonal agents.
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- 2020
21. The carbonate concentration mechanism of Pyropia yezoensis (Rhodophyta): evidence from transcriptomics and biochemical data
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Linwen He, Yuanyuan Sun, Xuehua Liu, Guangce Wang, Xiujun Xie, and Baoyu Zhang
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0106 biological sciences ,0301 basic medicine ,Carbonates ,Plant Science ,Photosynthesis ,01 natural sciences ,03 medical and health sciences ,Total inorganic carbon ,Gene Expression Regulation, Plant ,Malate synthase ,Carbonic anhydrase ,lcsh:Botany ,Carbon concentrating mechanism ,Enzyme activity ,biology ,Gene Expression Profiling ,Isocitrate lyase ,Seaweed ,Pyruvate carboxylase ,lcsh:QK1-989 ,030104 developmental biology ,Biochemistry ,Rhodophyta ,biology.protein ,Phosphoenolpyruvate carboxykinase ,Phosphoenolpyruvate carboxylase ,Pyropia yezoensis ,Transcriptome ,Photosynthetic efficiency ,Research Article ,010606 plant biology & botany - Abstract
Background Pyropia yezoensis (Rhodophyta) is widely cultivated in East Asia and plays important economic, ecological and research roles. Although inorganic carbon utilization of P. yezoensis has been investigated from a physiological aspect, the carbon concentration mechanism (CCM) of P. yezoensis remains unclear. To explore the CCM of P. yezoensis, especially during its different life stages, we tracked changes in the transcriptome, photosynthetic efficiency and in key enzyme activities under different inorganic carbon concentrations. Results Photosynthetic efficiency demonstrated that sporophytes were more sensitive to low carbon (LC) than gametophytes, with increased photosynthesis rate during both life stages under high carbon (HC) compared to normal carbon (NC) conditions. The amount of starch and number of plastoglobuli in cells corresponded with the growth reaction to different inorganic carbon (Ci) concentrations. We constructed 18 cDNA libraries from 18 samples (three biological replicates per Ci treatment at two life cycles stages) and sequenced these using the Illumina platform. De novo assembly generated 182,564 unigenes, including approximately 275 unigenes related to CCM. Most genes encoding internal carbonic anhydrase (CA) and bicarbonate transporters involved in the biophysical CCM pathway were induced under LC in comparison with NC, with transcript abundance of some PyCAs in gametophytes typically higher than that in sporophytes. We identified all key genes participating in the C4 pathway and showed that their RNA abundances changed with varying Ci conditions. High decarboxylating activity of PEPCKase and low PEPCase activity were observed in P. yezoensis. Activities of other key enzymes involved in the C4-like pathway were higher under HC than under the other two conditions. Pyruvate carboxylase (PYC) showed higher carboxylation activity than PEPC under these Ci conditions. Isocitrate lyase (ICL) showed high activity, but the activity of malate synthase (MS) was very low. Conclusion We elucidated the CCM of P. yezoensis from transcriptome and enzyme activity levels. All results indicated at least two types of CCM in P. yezoensis, one involving CA and an anion exchanger (transporter), and a second, C4-like pathway belonging to the PEPCK subtype. PYC may play the main carboxylation role in this C4-like pathway, which functions in both the sporophyte and gametophyte life cycles.
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- 2020
22. Structural and energetic understanding of novel natural inhibitors of Mycobacterium tuberculosis malate synthase
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Timir Tripathi, Rohit Shukla, and Harish Shukla
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0301 basic medicine ,biology ,Stereochemistry ,Chemistry ,Glyoxylate cycle ,Cell Biology ,Isocitrate lyase ,Metabolism ,biology.organism_classification ,Biochemistry ,Mycobacterium tuberculosis ,Citric acid cycle ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,Protein structure ,Docking (molecular) ,030220 oncology & carcinogenesis ,Malate synthase ,biology.protein ,Molecular Biology - Abstract
Persistent infection by Mycobacterium tuberculosis requires the glyoxylate shunt. This is a bypass to the tricarboxylic acid cycle in which isocitrate lyase (ICL) and malate synthase (MS) catalyze the net incorporation of carbon during mycobacterial growth on acetate or fatty acids as the primary carbon source. To identify a potential antitubercular compound, we performed a structure-based screening of natural compounds from the ZINC database (n = 1 67 740) against the M tuberculosis MS (MtbMS) structure. The ligands were screened against MtbMS, and 354 ligands were found to have better docking score. These compounds were assessed for Lipinski and absorption, distribution, metabolism, excretion, and toxicity prediction where 15 compounds were found to fit well for redocking studies. After refinement by molecular docking and drug-likeness analysis, four potential inhibitors (ZINC1483899, ZINC1754310, ZINC2269664, and ZINC15729522) were identified. These four ligands with phenyl-diketo acid were further subjected to molecular dynamics simulation to compare the dynamics and stability of the protein structure after ligand binding. The binding energy analysis was calculated to determine the intermolecular interactions. Our results suggested that the four compounds had a binding free energy of -201.96, -242.02, -187.03, and -169.02 kJ·mol-1 , for compounds with IDs ZINC1483899, ZINC1754310, ZINC2269664, and ZINC15729522, respectively. We concluded that two compounds (ZINC1483899 and ZINC1754310) displayed considerable structural and pharmacological properties and could be probable drug candidates to fight against M tuberculosis parasites.
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- 2018
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23. Lack of the NAD+-dependent glycerol 3-phosphate dehydrogenase impairs the function of transcription factors Sip4 and Cat8 required for ethanol utilization in Kluyveromyces lactis
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Montserrat Vega, Rosaura Rodicio, Fernando Moreno, Hans-Peter Schmitz, Jürgen J. Heinisch, and Lucía Mojardín
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0301 basic medicine ,Kluyveromyces lactis ,030102 biochemistry & molecular biology ,Glyoxylate cycle ,Dehydrogenase ,Isocitrate lyase ,Biology ,biology.organism_classification ,Microbiology ,03 medical and health sciences ,030104 developmental biology ,Glycerol-3-phosphate dehydrogenase ,Biochemistry ,Kluyveromyces ,Malate synthase ,Genetics ,biology.protein ,NAD+ kinase - Abstract
The NAD+-dependent glycerol 3-phosphate dehydrogenase (KlGpd1) is an important enzyme for maintenance of the cytosolic redox balance in the milk yeast Kluyveromyces lactis. The enzyme is localized in peroxisomes and in the cytosol, indicating its requirement for the oxidation of NADH in both compartments. Klgpd1 mutants grow more slowly on glucose than wild-type cells and do not grow on ethanol as a sole carbon source. We studied the molecular basis of the latter phenotype and found that Gpd1 is required for high expression of KlICL1 and KlMLS1 which encode the key enzymes of the glyoxylate pathway isocitrate lyase and malate synthase, respectively. This regulation is mediated by CSRE elements in the promoters of these genes and the Snf1-regulated transcription factors KlCat8 and KlSip4. To study the transactivation function of these factors we developed a modified yeast one-hybrid system for K. lactis, using the endogenous s-galactosidase gene LAC4 as a reporter in a lac9 deletion background. In combination with ChIP analyses we discovered that Gpd1 controls both the specific binding of Cat8 and Sip4 to the target promoters and the capacity of these factors to activate the reporter gene expression. We propose a model in which KlGpd1 activity is required for maintenance of the redox balance. In its absence, genes which function in generating redox balance instabilities are not expressed. A comparison of mutant phenotypes further indicates, that this system not only operates in K. lactis, but also in Saccharomyces cerevisiae.
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- 2018
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24. Monoterpenoid perillyl alcohol impairs metabolic flexibility of Candida albicans by inhibiting glyoxylate cycle
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Saif Hameed, Moiz A. Ansari, Kamal Ahmad, and Zeeshan Fatima
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0301 basic medicine ,Cell Survival ,030106 microbiology ,Biophysics ,Glyoxylate cycle ,Biochemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Bacterial Proteins ,Malate synthase ,Candida albicans ,Enzyme kinetics ,Molecular Biology ,chemistry.chemical_classification ,Dose-Response Relationship, Drug ,biology ,Chemistry ,Perillyl alcohol ,Malate Synthase ,Glyoxylates ,Gene Expression Regulation, Bacterial ,Cell Biology ,Isocitrate lyase ,biology.organism_classification ,Isocitrate Lyase ,Molecular biology ,Metabolic Flux Analysis ,Anti-Bacterial Agents ,Metabolic pathway ,030104 developmental biology ,Enzyme ,Monoterpenes ,biology.protein ,Metabolic Networks and Pathways - Abstract
The metabolic pathway such as glyoxylate cycle (GC) enables Candida albicans, to survive under glucose deficient conditions prevalent in the hostile niche. Thus its key enzymes (Isocitrate lyase; ICL and malate synthase; MLS) represent attractive targets against C. albicans. We have previously reported the antifungal potential of a natural monoterpenoid perillyl alcohol (PA). The present study uncovers additional role of PA as a potent GC inhibitor. We explored that PA phenocopied ICL1 deletion mutant and were hypersensitive under low carbon utilizing conditions. The effect of PA on GC was substantiated by molecular docking analyses, which reveals the in-silico binding affinity of PA with ICL and MLS and explored that PA binds to the active sites of both proteins with better binding energy in comparison to their known inhibitors 3-nitropropionate and bromopyruvate respectively. Enzyme kinetics by Lineweaver-Burk plot unravels that PA inhibits ICL and MLS enzymes in competitive and non-competitive manner respectively. Moreover, semi-quantitative RT-PCR indicated that PA inhibits ICL1 and MLS1 mRNA expressions. Lastly, we demonstrated the antifungal efficacy of PA by enhanced survival of Caenorhabditis elegans model and less hemolytic activity (10.6%) on human blood cells. Further studies are warranted for PA to be considered as viable drug candidate.
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- 2018
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25. A systems chemical biology study of malate synthase and isocitrate lyase inhibition in Mycobacterium tuberculosis during active and NRP growth.
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May, Elebeoba E., Leitão, Andrei, Tropsha, Alexander, and Oprea, Tudor I.
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SYSTEMS biology , *MALATE synthase , *ISOCITRATE lyase , *ENZYME inhibitors , *MYCOBACTERIUM tuberculosis , *MOLECULAR docking , *NATURAL resources - Abstract
Highlights: [•] SCB related virtual screening and docking studies can provide mechanistic insight on inhibition. [•] Unlike glyoxlate response, inhibitor impact on malate seems microenvironment dependent. [•] For similar microenvironments, ICL versus MS inhibition is metabolically more consequential. [•] Strong ICL inhibition causes metabolite levels in persistent Mtb toward levels in non-viable Mtb. [•] SCB points to a complex interplay between network structure, kinetics, and therapeutic outcome. [Copyright &y& Elsevier]
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- 2013
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26. Ability to grow on lipids accounts for the fully virulent phenotype in neutropenic mice of Aspergillus fumigatus null mutants in the key glyoxylate cycle enzymes
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Olivas, Israel, Royuela, Mar, Romero, Beatriz, Monteiro, M. Cândida, Mínguez, José M., Laborda, Fernando, and Lucas, J. Ramón De
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- *
ASPERGILLUS fumigatus , *LIPIDS , *NEUROSPORA crassa , *ANTIFUNGAL agents - Abstract
Abstract: Incidence and mortality rates of invasive aspergillosis clearly indicate the need of novel antifungals to treat patients suffering from this disease. Fungal proteins playing a crucial role in pathogenesis and with no orthologue in human cells are considered as primary therapeutic targets for the development of new antifungals with a high therapeutic index, one of the major drawbacks of the standard antifungal therapy, so far. In this work, we have analyzed the role in pathogenesis of the key enzymes of the Aspergillus fumigatus glyxoxylate cycle, isocitrate lyase and malate synthase, two possible candidates to primary therapeutic targets in this fungus. Deletion strains lacking isocitrate lyase (ΔacuD strains) or malate synthase (ΔacuE mutants) were constructed in this work. The Neurospora crassa pyr-4 gene was used as the replacing marker in gene deletion experiments. The pathogenicities of ΔacuD and ΔacuE mutants were tested in neutropenic mice and compared with those of two reference wild-type isolates A. fumigatus 237 and A. fumigatus 293. Interestingly, virulence and cytological studies clearly indicated the dispensability of the A. fumigatus glyoxylate cycle for pathogenicity. In addition, these results suggested the suitability of the pyr-4 gene as a valuable replacing marker for virulence studies in this fungus, a fact that was further confirmed by gene expression analyses. Finally, growth tests were performed to investigate the germination and growth of the ΔacuD and ΔacuE strains in nutrient deprivation environments, resembling the conditions that A. fumigatus conidia face after phagocytosis. Results obtained in this work strongly suggest that the ability to grow on lipids (triglycerides) of A. fumigatus isocitrate lyase and malate synthase deletion strains accounts for their fully virulent phenotype. [Copyright &y& Elsevier]
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- 2008
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27. Replacing the Ethylmalonyl-CoA Pathway with the Glyoxylate Shunt Provides Metabolic Flexibility in the Central Carbon Metabolism of Methylobacterium extorquens AM1
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Lennart Schada von Borzyskowski, Markus Buchhaupt, Jens Schrader, Julia A. Vorholt, Laura Pöschel, Frank Sonntag, and Tobias J. Erb
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0301 basic medicine ,Biomedical Engineering ,Glyoxylate cycle ,Heterologous ,Reductase ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Metabolic engineering ,03 medical and health sciences ,chemistry.chemical_compound ,Acyl-CoA Dehydrogenases ,Bacterial Proteins ,Methylmalonyl-CoA ,Methylobacterium extorquens ,Gene ,Acetic Acid ,biology ,Methanol ,Malate Synthase ,Glyoxylates ,General Medicine ,biology.organism_classification ,Formate Dehydrogenases ,Isocitrate Lyase ,Carbon ,Pyruvate carboxylase ,Alcohol Oxidoreductases ,030104 developmental biology ,Metabolic Engineering ,chemistry ,Biochemistry ,Spectrophotometry ,Crotonates ,Acyl Coenzyme A ,Oxidation-Reduction - Abstract
The ethylmalonyl-CoA pathway (EMCP) is an anaplerotic reaction sequence in the central carbon metabolism of numerous Proteo- and Actinobacteria. The pathway features several CoA-bound mono- and dicarboxylic acids that are of interest as platform chemicals for the chemical industry. The EMCP, however, is essential for growth on C1 and C2 carbon substrates and therefore cannot be simply interrupted to drain these intermediates. In this study, we aimed at reengineering central carbon metabolism of the Alphaproteobacterium Methylobacterium extorquens AM1 for the specific production of EMCP derivatives in the supernatant. Establishing a heterologous glyoxylate shunt in M. extorquens AM1 restored wild type-like growth in several EMCP knockout strains on defined minimal medium with acetate as carbon source. We further engineered one of these strains that carried a deletion of the gene encoding crotonyl-CoA carboxylase/reductase to demonstrate in a proof-of-concept the specific production of crotonic acid in the supernatant on a defined minimal medium. Our experiments demonstrate that it is in principle possible to further exploit the EMCP by establishing an alternative central carbon metabolic pathway in M. extorquens AM1, opening many possibilities for the biotechnological production of EMCP-derived compounds in future.
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- 2017
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28. Multiple pathways for acetate assimilation in Streptomyces cinnamonensis.
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Akopiants, Konstantin, Florova, Galina, Chaoxuan Li, and Reynolds, Kevin A.
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ISOCITRATE lyase , *STREPTOMYCES , *STREPTOMYCETACEAE , *LYASES , *MICROBIOLOGY , *ENZYMES - Abstract
In most bacteria acetate assimilation is accomplished via the glyoxylate pathway. Isocitrate lyase (ICL) and malate synthase (MS) are two key enzymes of this pathway, which results in the net generation of one molecule of succinyl-CoA from two acetyl-CoA molecules. Genetic and biochemical data have shown that genes encoding these key enzymes are present in streptomycetes, yet there has been no clear demonstration of the importance of these genes to acetate assimilation. In fact, for Streptomyces collinus an alternative butyryl-CoA pathway has been shown to be critical for growth on acetate as a sole carbon source. Crotonyl-CoA reductase (CCR) is a key enzyme in this pathway and catalyzes the last step of the conversion of 2-acetyl-CoA molecules to butyryl-CoA. In Streptomyces cinnamonensis C730.1, it has been shown that CCR and this butyryl-CoA pathway provide the majority of methylmalonyl-CoA and ethylmalonyl-CoA for monensin A biosynthesis in an oil-based fermentation medium. We have cloned a MS homologue gene from this strain. Reverse transcription and direct enzyme assays demonstrated that neither this nor other MS genes were expressed during fermentation in an oil-based fermentation of either the C730.1 or L1 strain (a ccr mutant). Similarly, no ICL activity could be detected. The C730.1 but not the L1 strain was able to grow on acetate as a sole carbon source. The Streptomyces coelicolor aceA and aceB2 genes encoding ICL and MS were cloned into a Streptomyces expression plasmid (a derivative of pSET152) to create pExIM1. Enzyme assays and transcript analyses demonstrated expression of both of these proteins in C730.1/pExIM1 and L1/pExIM1 grown in an oil-based fermentation and tryptic soy broth media. Nonetheless, L1/pExIM1, like L1, was unable to grow on acetate as a sole carbon source, and was unable to efficiently generate precursors for monensin A biosynthesis in an oil-based fermentation, indicating that the additional presence of these two enzyme activities does not permit a functional glyoxylate cycle to occur. UV mutagenesis of S. cinnamonensis L1 and L1/pExIM1 led to mutants which were able to grow efficiently on acetate despite a block in the butyryl-CoA pathway. Analysis of enzyme activity and monensin production from these mutants in an oil-based fermentation demonstrated that neither the glyoxylate cycle nor the butyryl-CoA pathway function, suggesting the possibility of alternative pathways of acetate assimilation. [ABSTRACT FROM AUTHOR]
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- 2006
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29. Activity and functional properties of the isocitrate lyase in the cyanobacterium Cyanothece sp. PCC 7424
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Henning Knoop, Marianne Gründel, and Ralf Steuer
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0301 basic medicine ,Cyanobacteria ,food.ingredient ,Photoperiod ,Cyanothece ,030106 microbiology ,Glyoxylate cycle ,Acetates ,Photosynthesis ,Microbiology ,03 medical and health sciences ,food ,Malate synthase ,Cell Proliferation ,biology ,Malate Synthase ,Glyoxylates ,Heterotrophic Processes ,Metabolism ,Isocitrate lyase ,biology.organism_classification ,Isocitrate Lyase ,Citric acid cycle ,Biochemistry ,biology.protein - Abstract
Cyanobacteria are ubiquitous photoautotrophs that assimilate atmospheric CO2 as their main source of carbon. Several cyanobacteria are known to be facultative heterotrophs that are able to grow on diverse carbon sources. For selected strains, assimilation of organic acids and mixotrophic growth on acetate has been reported for decades. However, evidence for the existence of a functional glyoxylate shunt in cyanobacteria has long been contradictory and unclear. Genes coding for isocitrate lyase (ICL) and malate synthase were recently identified in two strains of the genus Cyanothece, and the existence of the complete glyoxylate shunt was verified in a strain of Chlorogloeopsis fritschii. Here, we report that the gene PCC7424_4054 of the strain Cyanothece sp. PCC 7424 encodes an enzymatically active protein that catalyses the reaction of ICL, an enzyme that is specific for the glyoxylate shunt. We demonstrate that ICL activity is induced under alternating day/night cycles and acetate-supplemented cultures exhibit enhanced growth. In contrast, growth under constant light did not result in any detectable ICL activity or enhanced growth of acetate-supplemented cultures. Furthermore, our results indicate that, despite the presence of a glyoxylate shunt, acetate does not support continued heterotrophic growth and cell proliferation. The functional validation of the ICL is supplemented with a bioinformatics analysis of enzymes that co-occur with the glyoxylate shunt. We hypothesize that the glyoxylate shunt in Cyanothece sp. PCC 7424, and possibly other nitrogen-fixing cyanobacteria, is an adaptation to a specific ecological niche and supports assimilation of nitrogen or organic compounds during the night phase.
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- 2017
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30. Horticultural Production of Ultra High Resveratrol Peanut
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Jeneanne M. Kirven, Paul G. Johnson, Godson O. Osuji, Sela Woldesenbet, and Eustace Duffus
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0106 biological sciences ,chemistry.chemical_classification ,biology ,food and beverages ,Fatty acid ,General Medicine ,Isocitrate lyase ,Resveratrol ,Stilbenoid ,01 natural sciences ,Malate dehydrogenase ,chemistry.chemical_compound ,Biosynthesis ,chemistry ,Biochemistry ,010608 biotechnology ,Malate synthase ,biology.protein ,Phosphoenolpyruvate carboxylase ,010606 plant biology & botany - Abstract
Background: Resveratrol naturally occurring antioxidant in peanut (Legume: Arachis hypogaea) has phytochemical human health dietary effects associated with reduced inflammatory cancer risks. Its levels in peanut are ultra-low and variable (0 to 26 μg·g-1), which has made it difficult to market as a consistent high resveratrol produce. Objective: Understanding the regulation of resveratrol accumulation in peanut might lead to development of new techniques for optimizing and stabilizing its yield. Method: Peanuts were cultivated in horticultural field plots and treated with solutions of mineral salts (sulfate, potassium, phosphate, ammonium ion) that were optimized in stoichiometric (reactive) ratios. Peanut seed’s RNAs were subjected to Northern blot analysis for profiling the RNAs synthesized by glutamate dehydrogenase (GDH), and mRNAs encoding resveratrol synthase. The seed’s extracts were analyzed by GC-MS for determination of the resveratrol and fatty acid compositions. Result: Stoichiometric mixes of mineral ions induced the peanut GDH to synthesize some RNA that silenced the mRNAs encoding resveratrol synthase, phosphoglucomutase, isocitrate lyase, malate synthase, enolase, phosphoenolpyruvate carboxylase, malate dehydrogenase, and phosphoglycerate mutase in the control, KN-, and NPKS-treated but not in the NPPK-treated peanut. These resulted to decreased resveratrol content (6.0 μg·g-1) in the control peanut but maximized it (1.15 mg·g-1) in the NPPK-treated peanut. Therefore, resveratrol accumulation was optimized by coupling of glycolysis and citric-glyoxylic acid cycles to resveratrol biosynthesis. Fatty acid content of control (55.6 g·kg-1) was higher than the NPKS-treated (48.5 g·kg-1) and NPPK-treated peanut (44.9 g·kg-1) meaning that malonyl-CoA intermediate in both fatty acid and stilbenoid pathways was diverted to support maximum resveratrol biosynthesis in the NPPK-treated peanut. Conclusion: The functional coupling of citric-glyoxylic acid cycles and glycolysis to optimize resveratrol biosynthesis may encourage development of horticultural technology specific for production of ultra-high resveratrol peanuts.
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- 2017
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31. Molecular characterization of a bifunctional glyoxylate cycle enzyme, malate synthase/isocitrate lyase, in Euglena gracilis
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Nakazawa, Masami, Minami, Tomomi, Teramura, Koji, Kumamoto, Shohei, Hanato, Sayaka, Takenaka, Shigeo, Ueda, Mitsuhiro, Inui, Hiroshi, Nakano, Yoshihisa, and Miyatake, Kazutaka
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- *
EUGLENA gracilis , *ENZYMES , *ALCOHOL , *ISOCITRATE lyase , *TRANSFERASES , *CAENORHABDITIS elegans , *MOLECULAR evolution - Abstract
Abstract: Euglena gracilis induced glyoxylate cycle enzymes when ethanol was fed as a sole carbon source. We purified, cloned and characterized a bifunctional glyoxylate cycle enzyme from E. gracilis (EgGCE). This enzyme consists of an N-terminal malate synthase (MS) domain fused to a C-terminal isocitrate lyase (ICL) domain in a single polypeptide chain. This domain order is inverted compared to the bifunctional glyoxylate cycle enzyme in Caenorhabditis elegans, an N-terminal ICL domain fused to a C-terminal MS domain. Purified EgGCE catalyzed the sequential ICL and MS reactions. ICL activity of purified EgGCE increased in the existence of acetyl-CoA at a concentration of micro-molar order. We discussed the physiological roles of the bifunctional glyoxylate cycle enzyme in these organisms as well as its molecular evolution. [Copyright &y& Elsevier]
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- 2005
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32. Metal-catalyzed oxidation induces carbonylation of peroxisomal proteins and loss of enzymatic activities
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Nguyen, A.T. and Donaldson, R.P.
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PROTEINS , *OXIDATION , *DEHYDROGENASES , *GEL electrophoresis - Abstract
Abstract: Peroxisomes are involved in oxidative metabolic reactions and have the capacity to generate large amounts of reactive oxygen species that could damage biomolecules including their own resident proteins. The purpose of this study was to determine whether peroxisomal proteins are susceptible to oxidation and whether oxidative damage affects their enzymatic activity. Peroxisomal proteins were subjected to metal-catalyzed oxidation (MCO) with CuCl2/ascorbate and derivatized with 2,4-dinitrophenylhydrazine which allowed for spectrophotometric quantification of carbonylation. Immunochemical detection of carbonylated peroxisomal proteins, resolved by gel electrophoresis and detected with anti-DNP antibodies, revealed five oxidatively modified proteins with the following molecular weights: 80, 66, 62, 55, and 50kDa. The proteins at 66, 62, and 55kDa were identified as malate synthase (MS), isocitrate lyase, and catalase (CAT), respectively. MS and CAT were estimated to contain 2–3mol of carbonyl/mol of protein as a result of MCO. Enzymatic assays revealed varying degrees of activity loss. Isocitrate lyase and malate synthase showed significant loss of activity while catalase and malate dehydrogenase were less inhibited by carbonylation. Our findings show that peroxisomal proteins are vulnerable to MCO damage and thus may also be affected by in vivo exposure to reactive oxygen species. [Copyright &y& Elsevier]
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- 2005
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33. Anaerobic Induction of Isocitrate Lyase and Malate Synthase in Submerged Rice Seedlings Indicates the Important Metabolic Role of the Glyoxylate Cycle.
- Author
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Ying Lu, Yong-Rui Wu, and Bin Han
- Subjects
ISOCITRATE lyase ,KREBS cycle ,METABOLISM ,MESSENGER RNA ,LYASES ,GENES - Abstract
The glyoxylate cycle is a modified form of the tricarboxylic acid cycle that converts C2 compounds into C4 dicarboxylic acids at plant developmental stages. By studying submerged rice seedlings, we revealed the activation of the glyoxylate cycle by identifying the increased transcripts of mRNAs of the genes of isocitrate lyase (ICL) and malate synthase (MS), two characteristic enzymes of the glyoxylate cycle. Northern blot analysis showed that ICL and MS were activated in the prolonged anaerobic environment. The activity assay of pyruvate decarboxylase and ICL in the submerged seedlings indicated an 8.8-fold and 3.5-fold increase over that in the unsubmerged seedlings, respectively. The activity assay of acetyl-coenzyme A synthetase in the submerged seedlings indicated a 3-fold increase over that in the unsubmerged seedlings, which is important for initiating acetate metabolism. Consequently, we concluded that the glyoxylate cycle was involved in acetate metabolism under anaerobic conditions. Edited by: Hong-Xuan LIN [ABSTRACT FROM AUTHOR]
- Published
- 2005
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34. Endorsing functionality of Burkholderia pseudomallei glyoxylate cycle genes as anti-persistence drug screens
- Author
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Lye, Yu Min, Chan, Maurice, and Sim, Tiow-Suan
- Subjects
- *
ENZYMES , *CATALYSTS , *ENZYMOLOGY , *PHYSICAL & theoretical chemistry - Abstract
Abstract: Isocitrate lyase (ICL) and malate synthase (MS) are key glyoxylate cycle enzymes shown to be required for the persistence and virulence of Candida albicans and of Mycobacterium tuberculosis in macrophages because the up-regulation of glyoxylate genes and the corresponding enzymes could replenish C4 carbohydrates from C2 compounds in a persistent pathogen. In this study, the ace (acetate) genes (aceA and aceB) of a persistent pathogen, Burkholderia pseudomallei (ATCC 23343), encoding an ICL and a MS, respectively, were isolated and fully sequenced. The genes, aceA (1.3kb) and aceB (1.6kb) were cloned and expressed as tagged fusion proteins in Escherichia coli BL21 (DE3). The molecular weights of the predicted enzymes (ICL, 47.7kDa and MS, 59.1kDa) were consistent with ICLs and MSs reported so far. Phylogenetic analysis of these genes revealed significant identity (80–90%) with most bacterial ICLs and MSs. Comparative structural modeling and the localization of major ICL and MS family domains in the deduced peptide sequences showed interestingly significant similarity with isozymes from known pathogens. Specific activities of expressed ICL (589.27nmolmin−1 mg−1) and MS (485.54nmolmin−1 mg−1) were also demonstrated. Taken together, these results provide evidence for the functionality of glyoxylate cycle genes in B. pseudomallei and may thus be useful for designing antimicrobials targeted at the glyoxylate cycle. [Copyright &y& Elsevier]
- Published
- 2005
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35. Purification and Properties of Isocitrate Lyase from Pupas of the Butterfly Papilio machaon L.
- Author
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Eprintsev, A. T., Shevchenko, M. Yu., and Popov, V. N.
- Subjects
- *
ISOCITRATE lyase , *PUPAE , *PAPILIO , *BUTTERFLIES , *ENZYMES , *CHROMATOGRAPHIC analysis - Abstract
Key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, were identified in pupas of the butterfly Papilio machaon L. The activities of these enzymes in pupas were 0.056 and 0.108 unit per mg protein, respectively. Isocitrate lyase was purified by a combination of various chromatographic steps including ammonium sulfate fractionation, ion- exchange chromatography on DEAE-Toyopearl, and gel filtration. The specific activity of the purified enzyme was 5.5 units per mg protein, which corresponded to 98-fold purification and 6% yield. The enzyme followed Michaelis-Menten kinetics (Km for isocitrate, 1.4 mM) and was competitively inhibited by succinate (Ki = 1.8 mM) and malate (R = 1 mM). The study of physicochemical properties of the enzyme showed that it is a homodimer with a subunit molecular weight of 68 ± 2 kD and a pH optimum of 7.5 (in Tris-HCl buffer). [ABSTRACT FROM AUTHOR]
- Published
- 2004
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36. The glyoxylate bypass of Ralstonia eutropha
- Author
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Wang, Zheng-Xiang, Brämer, Christian O., and Steinbüchel, Alexander
- Subjects
- *
GENES , *RALSTONIA , *CLONING , *ESCHERICHIA coli - Abstract
The glyoxylate bypass genes aceA1 (isocitrate lyase 1, ICL1), aceA2 (isocitrate lyase 2, ICL2) and aceB1 (malate synthase, MS1) of Ralstonia eutropha HF39 were cloned, sequenced and functionally expressed in Escherichia coli. Interposon-mutants of all three genes (ΔaceA1, ΔaceA2 and ΔaceB1) were constructed, and the phenotypes of the respective mutants were investigated. Whereas R. eutropha HF39ΔaceA1 retained only 19% of ICL activity and failed to grow on acetate, R. eutropha HF39ΔaceA2 retained 84% of acetate-inducible ICL activity, and growth on acetate was not retarded. These data suggested that ICL1 is in contrast to ICL2 induced by acetate and specific for the glyoxylate cycle. R. eutropha HF39ΔaceB1 retained on acetate as well as on gluconate about 41–42% of MS activity and exhibited retarded growth on acetate, indicating the presence of a second hitherto not identified MS in R. eutropha HF39. Whereas in R. eutropha HF39ΔaceA1 and R. eutropha HF39ΔaceA2 the yields of poly(3-hydroxybutyric acid), using gluconate as carbon source, were significantly reduced, R. eutropha HF39ΔaceB1 accumulated the same amount of this polyester from gluconate as well as from acetate as R. eutropha HF39. [Copyright &y& Elsevier]
- Published
- 2003
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37. Two phenotypically compensating isocitrate dehydrogenases in Ralstonia eutropha
- Author
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Wang, Zheng-Xiang, Brämer, Christian, and Steinbüchel, Alexander
- Subjects
- *
RALSTONIA , *KREBS cycle , *ISOCITRATE lyase - Abstract
The tricarboxylic acid (TCA) cycle enzyme isocitrate dehydrogenase (IDH) and the glyoxylate bypass enzyme isocitrate lyase are involved in catabolism of isocitrate and play a key role in controlling the metabolic flux between the TCA cycle and the glyoxylate shunt. Two IDH genes icd1 and icd2 of Ralstonia eutropha HF39, encoding isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2), were identified and characterized. Icd1 was functionally expressed in Escherichia coli, whereas icd2 was expressed in E. coli but no activity was obtained. Interposon-mutants of icd1 (HF39Δicd1) and icd2 (HF39Δicd2) of R. eutropha HF39 were constructed and their phenotypes were investigated. HF39Δicd1 retained 43% of IDH activity, which was not induced by acetate, and HF39Δicd2 expressed 74% of acetate-induced IDH activity. Both HF39Δicd1and HF39Δicd2 kept the same growth rate on acetate as the wild-type. These data suggested that IDH1 is induced by acetate. The interposon-mutants HF39Δicd1 and HF39Δicd2 accumulated the same amount of poly(3-hydroxybutyric acid) as the wild-type. [Copyright &y& Elsevier]
- Published
- 2003
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38. Alternative fate of glyoxylate during acetate and hexadecane metabolism in Acinetobacter oleivorans DR1
- Author
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Chulwoo Park, Bora Shin, and Woojun Park
- Subjects
0301 basic medicine ,030106 microbiology ,Mutant ,Glyoxylate cycle ,lcsh:Medicine ,Acetates ,medicine.disease_cause ,Article ,03 medical and health sciences ,chemistry.chemical_compound ,Lactate dehydrogenase ,Malate synthase ,Alkanes ,medicine ,lcsh:Science ,Escherichia coli ,Multidisciplinary ,biology ,Acinetobacter ,Dose-Response Relationship, Drug ,Gene Expression Profiling ,lcsh:R ,Wild type ,Malate Synthase ,Glyoxylates ,Isocitrate lyase ,Metabolism ,Gene Expression Regulation, Bacterial ,Isocitrate Lyase ,030104 developmental biology ,Soil microbiology ,Bacterial genes ,Biochemistry ,chemistry ,Mutation ,biology.protein ,lcsh:Q - Abstract
The glyoxylate shunt (GS), involving isocitrate lyase (encoded by aceA) and malate synthase G (encoded by glcB), is known to play important roles under several conditions including oxidative stress, antibiotic defense, or certain carbon source metabolism (acetate and fatty acids). Comparative growth analyses of wild type (WT), aceA, and glcB null-strains revealed that aceA, but not glcB, is essential for cells to grow on either acetate (1%) or hexadecane (1%) in Acinetobacter oleivorans DR1. Interestingly. the aceA knockout strain was able to grow slower in 0.1% acetate than the parent strain. Northern Blot analysis showed that the expression of aceA was dependent on the concentration of acetate or H2O2, while glcB was constitutively expressed. Up-regulation of stress response-related genes and down-regulation of main carbon metabolism-participating genes in a ΔaceA mutant, compared to that in the parent strain, suggested that an ΔaceA mutant is susceptible to acetate toxicity, but grows slowly in 0.1% acetate. However, a ΔglcB mutant showed no growth defect in acetate or hexadecane and no susceptibility to H2O2, suggesting the presence of an alternative pathway to eliminate glyoxylate toxicity. A lactate dehydrogenase (LDH, encoded by a ldh) could possibly mediate the conversion from glyoxylate to oxalate based on our RNA-seq profiles. Oxalate production during hexadecane degradation and impaired growth of a ΔldhΔglcB double mutant in both acetate and hexadecane-supplemented media suggested that LDH is a potential detoxifying enzyme for glyoxylate. Our constructed LDH-overexpressing Escherichia coli strain also showed an important role of LDH under lactate, acetate, and glyoxylate metabolisms. The LDH-overexpressing E. coli strain, but not wild type strain, produced oxalate under glyoxylate condition. In conclusion, the GS is a main player, but alternative glyoxylate pathways exist during acetate and hexadecane metabolism in A. oleivorans DR1.
- Published
- 2019
39. A Universal Stress Protein That Controls Bacterial Stress Survival in Micrococcus luteus
- Author
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William R. Widger, Steven J. Bark, Preethi H. Gunaratne, Thinh D. Duong, Jonathan Rangel, Brandon Mistretta, Jesse Murphy, Jacob D. Storey, Rene Zimmerer, Abiodun Bodunrin, and Spencer Havis
- Subjects
Multidrug tolerance ,Citric Acid Cycle ,Glyoxylate cycle ,Biology ,medicine.disease_cause ,Microbiology ,03 medical and health sciences ,Bacterial Proteins ,Stress, Physiological ,Malate synthase ,medicine ,Molecular Biology ,Escherichia coli ,Heat-Shock Proteins ,030304 developmental biology ,0303 health sciences ,030306 microbiology ,Mycobacterium smegmatis ,fungi ,Glyoxylates ,Isocitrate lyase ,biology.organism_classification ,Micrococcus luteus ,biology.protein ,bacteria ,Bacteria ,Research Article - Abstract
Bacteria have remarkable mechanisms to survive severe external stresses, and one of the most enigmatic is the nonreplicative persistent (NRP) state. Practically, NRP bacteria are difficult to treat, and so inhibiting the proteins underlying this survival state may render such bacteria more susceptible to external stresses, including antibiotics. Unfortunately, we know little about the proteins and mechanisms conferring survival through the NRP state. Here, we report that a universal stress protein (Usp) is a primary regulator of bacterial survival through the NRP state in Micrococcus luteus NCTC 2665, a biosafety level 1 (BSL1) mycobacterial relative. Usps are widely conserved, and bacteria, including Mycobacterium tuberculosis, Mycobacterium smegmatis, and Escherichia coli, have multiple paralogs with overlapping functions that have obscured their functional roles. A kanamycin resistance cassette inserted into the M. luteus universal stress protein A 616 gene (ΔuspA616::kanM. luteus) ablates the UspA616 protein and drastically impairs M. luteus survival under even short-term starvation (survival, 83% wild type versus 32% ΔuspA616::kanM. luteus) and hypoxia (survival, 96% wild type versus 48% ΔuspA616::kanM. luteus). We observed no detrimental UspA616 knockout phenotype in logarithmic growth. Proteomics demonstrated statistically significant log-phase upregulation of glyoxylate pathway enzymes isocitrate lyase and malate synthase in ΔuspA616::kanM. luteus. We note that these enzymes and the M. tuberculosis UspA616 homolog (Rv2623) are important in M. tuberculosis virulence and chronic infection, suggesting that Usps are important stress proteins across diverse bacterial species. We propose that UspA616 is a metabolic switch that controls survival by regulating the glyoxylate shunt. IMPORTANCE Bacteria tolerate severe external stresses, including antibiotics, through a nonreplicative persistent (NRP) survival state, yet the proteins regulating this survival state are largely unknown. We show a specific universal stress protein (UspA616) controls the NRP state in Micrococcus luteus. Usps are widely conserved across bacteria, but their biological function(s) has remained elusive. UspA616 inactivation renders M. luteus susceptible to stress: bacteria die instead of adapting through the NRP state. UspA616 regulates malate synthase and isocitrate lyase, glyoxylate pathway enzymes important for chronic Mycobacterium tuberculosis infection. These data show that UspA616 regulates NRP stress survival in M. luteus and suggest a function for homologous proteins in other bacteria. Importantly, inhibitors of UspA616 and homologs may render NRP bacteria more susceptible to stresses, including current antibiotics.
- Published
- 2019
40. Progesterone Promotes Mitochondrial Respiration at the Biochemical and Molecular Level in Germinating Maize Seeds
- Author
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Hulya Turk
- Subjects
0106 biological sciences ,food.ingredient ,Plant Science ,progesterone ,maize ,01 natural sciences ,Article ,03 medical and health sciences ,food ,mitochondrial respiration ,Malate synthase ,Citrate synthase ,Cytochrome c oxidase ,Ecology, Evolution, Behavior and Systematics ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Ecology ,biology ,ATP synthase ,Chemistry ,Botany ,Isocitrate lyase ,Pyruvate dehydrogenase complex ,Enzyme ,germination ,Biochemistry ,QK1-989 ,gene expression ,biology.protein ,Cotyledon ,010606 plant biology & botany - Abstract
This research aimed to investigate the effects of progesterone, a mammalian steroid sex hormone, on the mitochondrial respiration in germinating maize seeds. For this purpose, maize seeds were divided into four different groups (control, 10−6, 10−8, and 10−10 mol·L−1 progesterone) and were grown in a germination cabinet in the dark at 24.5 ± 0.5 °C for 4 d. The changes in gene expression levels of citrate synthase (CS), cytochrome oxidase (COX19), pyruvate dehydrogenase (Pdh1), and ATP synthase (ATP6), which is involved in mitochondrial respiration, were studied in root and cotyledon tissues. Significant increases were recorded in the gene expression levels of all studied enzymes. In addition, progesterone applications stimulated activities of malate synthase (MS), isocitrate lyase (ICL), and alpha-amylase, which are important enzymes of the germination step. The changes in gene expression levels of mas1 and icl1 were found parallel to the rise in these enzymes’ activities. It was determined similar increases in root and coleoptile lengths and total soluble protein and total carbohydrate contents. The most remarkable changes were detected in 10−8 mol·L−1 progesterone-treated seedlings. These results clearly indicate that progesterone stimulates mitochondrial respiration by inducing biochemical and molecular parameters and thus accelerates seed germination thanks to the activation of other pathways related to mitochondrial respiration.
- Published
- 2021
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41. Mobilization of storage materials during light-induced germination of tomato ( Solanum lycopersicum ) seeds
- Author
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Dominika Jagiełło-Flasińska, Aleksandra Lewandowska, Aleksandra Eckstein, Klaus-J. Appenroth, Paweł Hermanowicz, and Halina Gabryś
- Subjects
0106 biological sciences ,0301 basic medicine ,Light ,Physiology ,Glyoxylate cycle ,Germination ,Plant Science ,seedling ,phytochromes ,01 natural sciences ,lipids ,03 medical and health sciences ,Phytochrome A ,Solanum lycopersicum ,Phytochrome B ,Malate synthase ,glyoxylate cycle ,Botany ,Genetics ,Plant Proteins ,Phytochrome ,biology ,starch ,Glyoxylates ,food and beverages ,Biological Transport ,Starch ,Isocitrate lyase ,biology.organism_classification ,Lipids ,red light ,030104 developmental biology ,Seedlings ,Seedling ,Mutation ,Seeds ,biology.protein ,Solanum ,010606 plant biology & botany - Abstract
The aim of this study was to analyze the metabolism of storage materials in germinating tomato (Solanum lycopersicum) seeds and to determine whether it is regulated by light via phytochromes. Wild type, single and multiple phytochrome A, B1 and B2 mutants were investigated. Imbibed seeds were briefly irradiated with far-red or far-red followed by red light, and germinated in darkness. Triacylglycerols and starch were quantified using biochemical assays in germinating seeds and seedlings during the first 5 days of growth. To investigate the process of fat-carbohydrate transformation, the activity of the glyoxylate cycle was assessed. Our results confirm the role of phytochrome in the control of tomato seed germination. Phytochromes A and B2 were shown to play specific roles, acting antagonistically in far-red light. While the breakdown of triacylglycerols proceeded independently of light, phytochrome control was visible in the next stages of the lipid-carbohydrate transformation. The key enzymes of the glyoxylate cycle, isocitrate lyase and malate synthase, were regulated by phytochrome(s). This was reflected in a greater increase of starch content during seedling growth in response to additional red light treatment. This study is the first attempt to build a comprehensive image of storage material metabolism regulation by light in germinating dicotyledonous seeds.
- Published
- 2016
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42. Peroxisomal microbodies are at the crossroads of acetate assimilation in the green microalga Chlamydomonas reinhardtii
- Author
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Kyle J. Lauersen, Marine Joris, Rémi Willamme, Nadine Coosemans, Olaf Kruse, and Claire Remacle
- Subjects
0106 biological sciences ,0301 basic medicine ,Glyoxylate cycle ,Chlamydomonas reinhardtii ,Isocitrate lyase ,Biology ,Peroxisome ,biology.organism_classification ,01 natural sciences ,Malate dehydrogenase ,03 medical and health sciences ,030104 developmental biology ,Biochemistry ,Malate synthase ,Glyoxysome ,biology.protein ,Microbody ,Agronomy and Crop Science ,010606 plant biology & botany - Abstract
The glyoxylate cycle is essential for growth on C2 compounds such as acetate. In this investigation, for the first time, we have elucidated the subcellular localization of the enzymes of the glyoxylate cycle in the green microalga Chlamydomonas reinhardtii. Acetyl-CoA synthase and malate dehydrogenase exist as multiple isoforms in this microalga, therefore, we first identified those implicated in the glyoxylate cycle based on the observation that lack of isocitrate lyase (ICL) in a previously identified icl deficient mutant was correlated with specific loss of the other enzymes of the glyoxylate cycle. In this work, we determined that five of the six enzymes associated with the glyoxylate cycle were found to be within peroxisomal microbodies. Citrate synthase, aconitase, malate synthase, malate dehydrogenase, and acetyl-CoA synthase are located in peroxisomal microbodies while isocitrate lyase is cytosolic. Our findings implicate a key role for these cellular compartments in acetate assimilation for Chlamydomonas. Microbodies have only recently been discovered in C. reinhardtii and their existence had been previously debated. The isoform specific subcellular localization determined here suggests that peroxisomal microbodies should be considered in the design of metabolic models for carbon assimilation in C. reinhardtii.
- Published
- 2016
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43. Role of Glyoxylate Shunt in Oxidative Stress Response
- Author
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Woojun Park, Eugene L. Madsen, Jaejoon Jung, In Ae Jang, and Sung-Eun Ahn
- Subjects
0301 basic medicine ,030106 microbiology ,Glyoxylate cycle ,Oxidative phosphorylation ,Acetates ,medicine.disease_cause ,Microbiology ,Biochemistry ,03 medical and health sciences ,Oxygen Consumption ,Malate synthase ,Aerobic denitrification ,medicine ,Molecular Biology ,biology ,Malate Synthase ,Computational Biology ,Glyoxylates ,Gene Expression Regulation, Bacterial ,Cell Biology ,Isocitrate lyase ,Isocitrate Lyase ,Citric acid cycle ,Oxidative Stress ,Metabolic pathway ,Biofilms ,Pseudomonas aeruginosa ,biology.protein ,Transcriptome ,Genome, Bacterial ,Metabolic Networks and Pathways ,Oxidative stress - Abstract
The glyoxylate shunt (GS) is a two-step metabolic pathway (isocitrate lyase, aceA; and malate synthase, glcB) that serves as an alternative to the tricarboxylic acid cycle. The GS bypasses the carbon dioxide-producing steps of the tricarboxylic acid cycle and is essential for acetate and fatty acid metabolism in bacteria. GS can be up-regulated under conditions of oxidative stress, antibiotic stress, and host infection, which implies that it plays important but poorly explored roles in stress defense and pathogenesis. In many bacterial species, including Pseudomonas aeruginosa, aceA and glcB are not in an operon, unlike in Escherichia coli. In P. aeruginosa, we explored relationships between GS genes and growth, transcription profiles, and biofilm formation. Contrary to our expectations, deletion of aceA in P. aeruginosa improved cell growth under conditions of oxidative and antibiotic stress. Transcriptome data suggested that aceA mutants underwent a metabolic shift toward aerobic denitrification; this was supported by additional evidence, including up-regulation of denitrification-related genes, decreased oxygen consumption without lowering ATP yield, increased production of denitrification intermediates (NO and N2O), and increased cyanide resistance. The aceA mutants also produced a thicker exopolysaccharide layer; that is, a phenotype consistent with aerobic denitrification. A bioinformatic survey across known bacterial genomes showed that only microorganisms capable of aerobic metabolism possess the glyoxylate shunt. This trend is consistent with the hypothesis that the GS plays a previously unrecognized role in allowing bacteria to tolerate oxidative stress.
- Published
- 2016
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44. Characterisation and structural analysis of glyoxylate cycle enzymes of Teladorsagia circumcincta
- Author
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Nikola Palevich, Charlotte L.G. Bouchet, Saleh Umair, and Heather V. Simpson
- Subjects
Models, Molecular ,Protein Conformation ,030231 tropical medicine ,Glyoxylate cycle ,medicine.disease_cause ,law.invention ,Structure-Activity Relationship ,03 medical and health sciences ,0302 clinical medicine ,law ,Malate synthase ,parasitic diseases ,medicine ,Animals ,Amino Acid Sequence ,Molecular Biology ,Escherichia coli ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Trichostrongyloidea ,biology ,Malate Synthase ,Glyoxylates ,Helminth Proteins ,Isocitrate lyase ,Hydrogen-Ion Concentration ,Recombinant Proteins ,Teladorsagia circumcincta ,Amino acid ,Enzyme Activation ,Molecular Weight ,Enzyme ,chemistry ,Biochemistry ,biology.protein ,Recombinant DNA ,Parasitology - Abstract
A 1332 bp full length cDNA encoding Teladorsagia circumcincta isocitrate lyase (TciICL) and a 1575 bp full length cDNA encoding T. circumcincta malate synthase (TciMS) were cloned, expressed in Escherichia coli and the recombinant proteins purified. The predicted TciICL protein of 444 amino acids was present as a single band of about 52 kDa on SDS-PAGE and the recombinant TciMS of 525 amino acids formed a single band about 62 kDa. Multiple alignments of the combined bifunctional TciICL MS protein sequence with homologues from other nematodes showed that the greatest similarity (89-92%) to the homologues of Ancylostoma ceylanicum, Haemonchus contortus and Haemonchus placei and 71-87% similarity to the other nematode sequences. The 3-dimensional structures, binding and catalytic sites were determined for TciICL and TciMS and shown to be highly conserved. Substrate and metal ion binding sites were identified and were completely conserved in other homologues. TciICL was confirmed as a functional enzyme. At 30 °C, the optimum pH was pH 7.5, the Vmax was 275 ± 23 nmoles.min-1.mg-1 protein and the apparent Km for the substrate isocitrate was 0.7 ± 0.01μM (mean ± SEM, n = 3). Addition of 10 mM metal ions (except Mg2+) or 1 mM inhibitors reduced the recombinant TciICL activity by 60-90%. Antibodies in both serum and saliva from field-immune, but not nematode-naïve, sheep recognised recombinant TciICL in ELISA, supporting similar antigenicity to that of the native enzyme.
- Published
- 2020
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45. Inhibition of Germination Gene Expression by Viviparous-1 and ABA during Maize Kernel Development.
- Author
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Nam Chon Paek, Lee, Byung-Moo, Dong Gyu Bai, and Smith, James D.
- Abstract
Two maize glyoxysomal genes expressed during germination, malate synthase (MS) and isocitrate lyase (ICL), were used to characterize the regulatory roles of the Viviparous-l (Vpl) regulatory gene and abscisic aicd (ABA) in the induction of embryo quiescence during kernel development. In wild-type maize embryo, MS and ICL transcripts were first detected at 2 (MS) or 3 (ICL) days after germination (DAG), peaked at 5 DAG, and decreased thereafter. By reverse transcriptasepolymerase chain reaction (RT-PCR), the germinationspecific genes were amplified in both ABA-insensitive (vpl) and ABA-deficient (vp7 and vplO) mutant embryos at 26 and 33 days after pollination (DAP), but not in wild-type embryos. The repression of these germination-specific genes thus r equires the Vpl gene product and normal levels of ABA to induce embryo quiescence during kernel development. This suggests that a genetic regulatory system exists to prevent vivipary in developing maize embryos. The involvement of the Vpl gene product and ABA in repressing germination-specific genes complements their previously defined roles in the induction of seed-specific genes such as Cl (Hattori et al., 1992; Paek et aI. , 1997), Em (McCarty et aI., 1991), and Glnl (Kriz et aI., 1990; Rivin and Grudt, 1991). [ABSTRACT FROM AUTHOR]
- Published
- 1998
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46. Influence of cytokinins on growth of Phycomyces blakesleeanus and on the activities of the glyoxylate cycle enzymes isocitrate lyase and malate synthase.
- Author
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Botz, Thomas and Hilgenberg, Willy
- Subjects
- *
PHYCOMYCES blakesleeanus , *CYTOKININS , *PLANT hormones , *CARBON , *OLEIC acid , *CELL culture - Abstract
Treatment of the 1 + strain of Phycomyces blakesleeanus Bgff. with various cytokinins resulted in a stimulation of growth. The magnitude of growth stimulation depended on both the structure of the hormone used and the carbon source in the culture medium. Most of the cytokinin derivatives were active effect in glucose and oleic acid cultures. Benzyladenine (BA) and benzyladenosine stimulated the fungal growth only when oleic add was the sole carbon source, while they had no effect in glucose cultures within the tested range of concentrations. [14C]-BA was accumulated by the mycelium of oleic acid cultures. Therefore, differences in BA uptake between glucose and oleic acid cultures could account mainly for the specific growth-promoting effect of BA. In oleic acid cultures isocitrate lyase (EC 4.1 3.1) and malate synthase (EC 4.1.3.2) activities were enhanced by 40 and 34%, respectively, in the presence of BA. A time course of the hormone effect suggests that BA is not involved in induction, but in the regulation of the mentioned enzymes in Phycocmyces. In contrast, acetate when presented as the sole carbon source or after addition to a glucose culture medium, induced isocitrate lyase activity. This enzyme induction was prevented by simultaneous addition of cycloheximide. [ABSTRACT FROM AUTHOR]
- Published
- 1987
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47. Characterization of Aspergillus nidulans peroxisomes by immunoelectron microscopy.
- Author
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Valenciano, Susana, De Lucas, J. R., Van der Klei, I., Veenhuis, Marten, and Laborda, F.
- Abstract
In previous work, we have demonstrated that oleate induces a massive proliferation of microbodies (peroxisomes) in Aspergillus nidulans. Although at a lower level, proliferation of peroxisomes also occurrs in cells growing under conditions that induce penicillin biosynthesis. Here, microbodies in oleate-grown A. nidulans cells were characterized by using several antibodies that recognize peroxisomal enzymes and peroxins in a broad spectrum of eukaryotic organisms such as yeast, and plant, and mammalian cells. Peroxisomes were immunolabeled by anti-SKL and anti-thiolase antibodies, which suggests that A. nidulans conserves both PTS1 and PTS2 import mechanisms. Isocitrate lyase and malate synthase, the two key enzymes of the glyoxylate cycle, were also localized in these organelles. In contrast to reports of Neurospora crassa, our results demonstrate that A. nidulans contains only one type of microbody (peroxisomes) that carry out the glyoxylate cycle and contain 3-ketoacyl-CoA thiolase and proteins with the C-terminal SKL tripeptide. [ABSTRACT FROM AUTHOR]
- Published
- 1998
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48. Regulation of acetate metabolism in Corynebacterium glutamicum: transcriptional control of the isocitrate lyase and malate synthase genes.
- Author
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Wendisch, Volker F., Spies, Marion, Reinscheid, D. J., Schnicke, Stephanie, Sahm, Hermann, and Eikmanns, B. J.
- Abstract
In the amino-acid-producing microorganism Corynebacterium glutamicum, the specific activities of the acetate-activating enzymes acetate kinase and phosphotransacetylase and those of the glyoxylate cycle enzymes isocitrate lyase and malate synthase were found to be high when the cells were grown on acetate (0.8, 2.9, 2.1, and 1.8 U/mg protein, respectively). When the cells were grown on glucose or on other carbon sources such as lactate, succinate, or glutamate, the specific activities were two- to fourfold (acetate kinase and phosphotransacetylase) and 45- to 100-fold (isocitrate lyase and malate synthase) lower, indicating that the synthesis of the four enzymes is regulated by acetate in the growth medium. A comparative Northern (RNA) analysis of the C. glutamicum isocitrate lyase and malate synthase genes ( aceA and aceB) and transcriptional cat fusion experiments revealed that aceA and aceB are transcribed as 1.6- and 2.7-kb monocistronic messages, respectively, and that the regulation of isocitrate lyase and malate synthase synthesis is exerted at the level of transcription from the respective promoters. Surprisingly, C. glutamicum mutants defective in either acetate kinase or phosphotransacetylase showed low specific activities of the other three enzymes (phosphotransacetylase, isocitrate lyase, and malate synthase or acetate kinase, isocitrate lyase, and malate synthase, respectively) irrespective of the presence or absence of acetate in the medium. This result and a correlation of a high intracellular acetyl coenzyme A concentration with high specific activities of isocitrate lyase, malate synthase, acetate kinase, and phosphotransacetylase suggest that acetyl coenzyme A or a derivative thereof may be a physiological trigger for the genetic regulation of enzymes involved in acetate metabolism of C. glutamicum. [ABSTRACT FROM AUTHOR]
- Published
- 1997
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49. Development and regulation of three glyoxysomal enzymes during cotton seed maturation and growth.
- Author
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Turley, Rickie and Trelease, Richard
- Abstract
The temporal, nonconcerted development of activities of malate synthase (MS), isocitrate lyase (ICL), and catalase (Cat) was explored in more detail in maturing and germinated cotton ( Gossypium hirsutum L.) seeds. RNA was extracted at six intervals beginning at 17 days post anthesis (DPA) through 72 hours post imbibition (HPI). In vitro translations revealed that mRNAs for each enzyme were translatable at all intervals. Enzyme activities and immunoselected proteins also were found at all intervals. Similar specific activities throughout maturation indicated that embryo cells were not accumulating inactive protein. The steady-state level of mRNAs encoding each enzyme exhibited different patterns of change during seed maturation, and each peaked at least 24 h before peak enzyme activities in germinated seeds. All three enzymes occur together as early as 17 DPA in a coordinate manner; however, the subsequent, nonconcerted increases in protein, activity, and mRNA for each enzyme indicate that developmental expression in cotton seed embryos is regulated in a noncoordinate fashion by as yet unidentified specific control mechanism(s). [ABSTRACT FROM AUTHOR]
- Published
- 1990
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50. Control of enzyme activities in cotton cotyledons during maturation and germination.
- Author
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Choinski, John, Trelease, Richard, and Doman, Diane
- Abstract
Cotton ( Gossypium hirsutum L.) embryos excised from bolls 38-43 d after anthesis and cultured in vitro for 4 d on a nutrient agar medium containing 3.8 μM abscisic acid (ABA) developed enzyme activity and accumulated insoluble protein, neutral lipid, and dry weight similar to embryos maturing on the plant. Inclusion of ABA in the medium prevented precosious germination and allowed continued increases in catalase, malate dehydrogenase, citrate synthase, aspartate aminotransferase, and β-oxidation enzyme activities as well as de-novo synthesis of malate synthase. Isocitrate lyase activity was not detectable in ABA-cultured embryos nor normally-developed embryos. Omission of sucrose from the medium resulted in near-doubling of the development of malate synthase activity, with minimal effects on the other enzyme activities. Addition of Actinomycin D, cordycepin, or cycloheximide to ABA-containing cultures did not overcome the observed inhibition of germination, but severely reduced both the appearance of new malate synthase activity and further production of other related enzyme activities. Thus, development of these enzyme activities in the presence of ABA appears dependent on transcription and translation, while inhibition of germination by ABA at this stage of development is not sensitive to the RNA- and protein-synthesis inhibitors. The results indicate that ABA does not prevent vivipary by suppressing translation of m-RNAs coding for isocitrate lyase and its companion enzymes, as previously proposed. [ABSTRACT FROM AUTHOR]
- Published
- 1981
- Full Text
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